Vera Mayhew and Brian Poole, Microbiology and Molecular Biology
Introduction
Epstein-Barr Virus (EBV) is one of the viruses found most frequently in the human population. Approximately 95% of the population is infected in the United Sates by the age of 40. When primary infection occurs is young adulthood approximately 50% of cases result in infectious mononucleosis. Whether EBV infection results in mononucleosis manifestations or not, the virus stays latent in the infected individual’s B cells for the rest of their life.
Systemic Lupus Erythematosus (SLE) is an autoimmune disease where the body begins to generate antibodies targeting nuclear antigens of the body’s own cells. Thus, when cells undergo normal programmed cell death, or if cell death occurs due to physical damage, an inflammatory reaction is triggered, causing further tissue damage and severe discomfort. SLE patients have been found to have higher EBV viral load and abnormal levels of immune cells (1, 2), and to be at a higher risk for certain lymphomas and cancers (3), strongly suggesting that EBV plays a role in lupus development and severity. EBV viral load has been found to substantially increase during lupus autoimmune flares, further strengthening this argument (4).
In order to perform experiments to investigate the role that EBV may play in SLE pathology, stable lymphoblastoid cell lines (LCL) must be established. The goal of this project was to generate SLE-positive and SLE-negative LCLs, and to investigate the cause behind why SLEpositive LCLs are harder to establish than control lines.
Methodology
Lymphocyte separation
Blood samples were collected from SLE patience as well as race- and sex-matched controls. Lymphocytes were extracted from samples using lymphocyte separation media. Isolated lymphocytes were cultured in RPMI 1640 media and incubated at 37 ℃.
Lymphoblastoid Cell Line (LCL) Generation
Lymphocytes isolated from patient samples were infected with Epstein-Barr virus (EBV) in order to immortalize the B cells. Cells were incubated and undisturbed for three weeks post infection in a T25 flask, and then transferred to a T75 flask and grown in fresh growth media till sufficient cell concentrations were achieved to freeze down cells for cryogenic storage. After initial infection, cyclosporin was added to growth media at a concentration of 2 μg/mL to keep T cells from attacking and killing infected B cells.
Soft-agar colony formation assay
Twenty-four hours post EBV infection, lymphocytes were plated in soft agar at one million cells per well in a 6-well plate in order to observe colony formation as infected B cells underwent clonal expansion. A 1 mL layer of 0.5% agarose containing the cells was poured on top of a 1 mL 0.5% agarose base layer. A 1mL layer of growth media was added on top of agar layers in order to keep the well hydrated and supplied with sufficient nutrients. Colony formation and cell death was observed and documented once a week, for three consecutive weeks, by taking pictures of each well using a microscope. Each cell line was run in triplicate.
Results
All cell lines from both SLE patients and controls failed to show colony formation in the softagar assays. No visible colonies were observed under the microscope. However, as expected, significant cell death was observed over time, which can be attributed to the non-B cell lymphocytes dying over time, as EBV does not infect these cells.
None of the SLE cells lines (n=6) were able to be successively transformed into a stable cell line. Two of the five control samples were successfully transformed into a stable lymphoblastoid cell line. These results support the observation made by other institutions and researchers that SLE cells have a significantly lower success rate when attempting to generate LCLs.
Discussion
Lymphoblastoid cell lines (LCLs) are immortalized B cell lines in which the B cells have been infected with Epstein-Barr virus so that they grow and divide indefinitely. When the lymphocytes are isolated from blood samples and treated with EBV, B cells are the only cell type that can be infected. These infected cells then proliferate and constitute the stable cell line. Researchers have previously observed that it is much more difficult to successfully establish a stable LCL from cell obtained from SLE patients. This observation was supported by the results of this study as well, as only non-SLE cells were successfully transformed. One explanation of this phenomenon is that SLE patient samples have lower numbers of cells that initially get infected and proliferate. In order to test this hypothesis, the EBV-treated cells were plated in a colony-formation assay that would immobilize the cells and allow colonies formed by proliferating cells to be counted. A new method, based off of colony-formation assays used for tumor cells, was designed and tested to see if it could be used as a more simple alternative to the standard colony-formation assay, which involves a more complicated layering of several cell types and growth mediums. The results show that this new method does not allow sufficient proliferation of the B cells. However, based on this result the lab has gained valuable knowledge that can save time if colony-formation need be measured in the future. Although the cause of the lower success rate could not be determined and SLE-positive LCLs could not be generated, the lab now has two control cell lines that will be valuable in future experiments.
Conclusion
While SLE-positive cell lines could not be successfully generated, and differences in colony formation as compared to SLE-negative cells was unable to be measured, the project yielded valuable insights into techniques that can be used for future projects.
- Draborg A. H., Duus K., and Houen G. 2012. Epstein-Barr virus and systemic lupus erythematosus. Clin Dev Immunol vol. 2012: 10 pages.
- Poole B. D., Templeton A. K., Guthridge J. M., Brown E. J., Harley J. B., James J. A. 2009. Aberrant Epstein–Barr viral infection in systemic lupus erythematosus. Autoimmun Rev 8(4):337-342.
- Bernatsky S., et al. 2013. Cancer risk in systemic lupus: an updated international multicentre cohort study. J Autoimmun 42:130-135.
- Martin L., et al. 2011. Exausted cytotoxic control of Epstein-Barr virus in human lupus. PLoS Pathog 7(10)